>When do birds sing?
Twilight. dawn and dusk. during the transitions.
"Elon Musk, from 50% to 90% of Twitter accounts for daily posts may in fact be Bots" found on glp
"He was listed as anattendee at subterranean challenge proposers day, an event held by The Defense Advanced Research Projects Agency, an agency of the United States Department of Defense.
He belonged a group ofLiberian veterans in the United Stateson Facebook."
"When asked why he needed so many high-power weapons, Bradley is accused of telling authorities that he works for a “secret government agency” and required the weaponry “for his mission,” according to The Boston Globe.
The Globe noted that Bradley had a valid gun permit in Texas but not Massachusetts. “He also stated that we could take the guns and store them at the Tewksbury Police Department, but he would have to come get them if he was called for his mission,” a police report said. “Francho went on to say that he can’t tell us what he does for work or why he has all the guns with him, but that he is down in this area working for a government agency that is dealing with avirus.”"
https://heavy.com/news/2018/03/francho-bradley-adrianne-jennings-gun-march/
"Look into Transfection with cDNA/mRNA. Bio lab garbage is a distraction. Keeps everyone looking at where it was created and not the technology of this mRNA."
https://onlinelibrary.wiley.com/doi/10.1002/biot.201900198
mRNA Transfection into CHO-Cells Reveals Production Bottlenecks
Abstract
Obtaining highly productive Chinese hamster ovary (CHO)-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbor potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product may therefore prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. The aim of the study is to reach specific productivities approaching those of industrial production cell lines by transfection of in vitro transcribed mRNA. The system is characterized with respect to transfection efficacy (by quantitative PCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturates at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remain linearly correlated to the amount of mRNA taken up. Nevertheless, MS shows a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization.
Abstract
Cell line development for the production of biotherapeutics is a time-consuming process, with the host cell-line having a high influence on the resulting product quality. Dose-dependent transfection of in vitro transcribed mRNA into CHO-K1 host cells reveals bottlenecks in protein-synthesis and posttranslational modifications. The presented method allows the researcher to subject any host cell-line of choice to a high specific productivity, allowing for a rapid screen of host cell to product compatibility, and insight into bottlenecks that may appear later on during cell line development.
4 Concluding Remarks
In summary, these experiments offer a proof-of-concept for using mRNA transfection to overload the cellular production machinery and detect cell and protein specific bottlenecks at high productivity. We could show that the levels of intracellular eGFP saturated and the quality of N-glycosylation of EPO-Fc decreased. Since these experiments were performed in rich medium within a short time frame, these changes most likely were not caused by media depletion, but rather by bottlenecks within the cellular protein synthesis and processing machinery in the ER and Golgi, or possibly by limited precursor availability within the cells. These results correspond well to changes in glycan quality observed in high producers of mAbs that had been shifted to higher productivity.23-25 The elucidation of the relationship of qP and N-glycan processing in these studies was limited by the range of qP achievable through bioprocess-modification alone. Our approach on the other hand covered a broad range of qP from 0.8 pg cell–1 d–1 to over 15 pg cell–1 d–1, and was able to show a dose-dependent decline in N-glycan processing of EPO-Fc. In particular, this range of qP is achieved within a day of transfection, as opposed to the months needed to generate high-producing clones.40
Optimization of mRNA stability, for example, by inclusion of modified nucleotides and alternate cap structures, or mRNA-circularization, in conjunction with repeated transfections, could yield even higher qP, observation of long-term adaptation to high productivities or limitations at a cell pool level, avoiding biases introduced by clonal effects. A more detailed analysis of cellular parameters, such as UPR-response41 or polysome profiling42 will be necessary, to see to which degree this method is able to recapitulate the physiological state of stably generated high producer clones.
Put into practice, this method could improve time- and cost-efficiency by I) enabling quicker analysis of the compatibility of protein and host cell line,43 II) allowing screens for a balance of high productivity and full processing for individual biotherapeutics, and III) indicating suitable targeted cell-line engineering approaches44, 45 early on during cell line development. The method offers an unprecedented dynamic range of production loads, as well as high degrees of flexibility with respect to both product and cell line.
https://www.jove.com/t/59626/in-vitro-transcribed-rna-based-luciferase-reporter-assay-to-study
Immunology and Infection
In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells
We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5’-untranslated region (UTR) and 3’-UTR. Different translation initiation modes can also be examined using this method.
Abstract
Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. To dissect the role of 5'-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5'-UTR and 3'-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006602
The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation
Pragyesh Dhungel,Shuai Cao,Zhilong Yang
Version 2
Published: August 30, 2017
Abstract
The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.
Could the 5’-poly(A) leader mediate cap-independent translation during poxvirus infection?
Many RNA viruses, such as piconaviruses, crucifer-infecting tobamovirus, hepatitis C virus, and Foot-and-mouth disease virus can synthesize their proteins through a cap-independent translation mode [41, 43–45]. The most studied mechanism is through viral IRESs, which usually bear highly complex structures to recruit 40S ribosome [46]. Some RNA viruses use other cap-independent mechanism such as 3’ cap-independent translational enhancer (3’CITE) in their mRNAs to recruit ribosome subunits [47, 48]. Cap-independent translation is less appreciated in DNA viruses. In this study, we show that a poly(A)-headed mRNA is efficiently translated in cells with impaired cap-dependent translation (Fig 8) as well as without an m7G cap (Fig 7), which strongly suggests that a short, unstructured 5’-poly(A) leader may mediate cap-independent translation in VACV-infected cells. In literature, Mulder et al. showed that VACV protein synthesis only requires a low level of intact translation initiation factor eIF4F [49]. In another in vitro study, Shirokikh et al. showed that the translation initiation complex could be formed on a 5’-poly(A) leader mRNA without the need of eIF4E, a rate-limiting and cap binding translation factor [50]. Additionally, in yeast, crucifer-infecting tobamovirus, and avian herpesvirus, an A-rich tract in some 5’-UTRs is suggested to function as an IRES [26, 41, 42], an RNA element allowing for a form of 5’ cap-independent translation initiation. Recruitment of poly(A) binding protein (PABP) through the A-rich tracts plays an important role in IRES-mediated cap-independent translation initiation. During the review process of this manuscript, Jha et al. reported that a small ribosomal subunit protein, receptor for activated C kinase (RACK1), is important for efficient translation of VACV post-replicative mRNAs [51]. RACK1 was previously found to be important for IRES-mediated cap-independent translation of several RNA viruses [52]. These findings support the possibility that the poly(A) can mediate cap-independent translation although it does not serve as an IRES (Fig 9).
https://www.yahoo.com/entertainment/first-human-patient-injected-revolutionary-140900099.html
First human patient injected with revolutionary cancer-killing virus
Scientists have injected the first human patient with a new cancer-killing virus. The virus, known as Vaxinia, has seen successful tests in animals. However, the true test of its efficacy begins with this new clinical trial.
It’s easy to hear the word virus and instantly think of something bad. After all, there are a lot of deadly viruses out there. However, scientists are using a new cancer-killing virus known as Vaxinia in an experimental cancer treatment.
The hope here is that the virus will amplify the body’s immune response against cancer. The virus itself has been engineered specifically to kill cancer cells. And, in previous animal trials, scientists have seen very promising results. These kinds of viruses have been a “smoking gun” in the fight against cancer for over a century.
However, the success of these viruses has been very limited, to say the least. This time around, though, the scientists have engineered the cancer-killing virus to not only harm cancer cells, but also to make them more recognizable to the body’s immune system. Researchers hope that this will help make the body’s response stronger, allowing it to fight back better.
Previous clinical trials of cancer drugs have shown promising results, too. But, Vaxinia could help open new doors, too.
Of course, before moving on to the human tests, the scientists tested the virus on animals. In many cases, they saw huge success at shrinking tumors in early animal and lab experiments. The cancer-killing virus has shown that it can reduce the size of lung, breast, ovarian, pancreatic, and colon cancer tumors.
With that success, the scientists decided to move on to human testing. Results seen in animals do not always directly translate to human patients. There are a lot of reasons for this, obviously, but the researchers are hopeful this virus could improve patients’ chance to fight back against cancer.
Currently, Vaxinia will be tested in a Phase 1 trial of just 100 cancer patients. These patients have metastatic or advanced solid tumors, and each has tried at least two other treatments. The researchers plan to administer the drug in two different groups. The first will receive just Vaxinia. The second group will get the cancer-killing virus plus an immunotherapy drug.
Of course, Phase 1 trials are mostly about safety and finding the optimal dose. As such, it might not prove the efficacy of the virus as a whole. However, it is an important next step in possibly finding an alternative method to fighting cancer. The trial is currently expected to complete by early 2025. So, it’ll be a while before we see any final results.
01/21/2022 10:43 PM by an AC:
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Everyone who has been injected with the (vaccine) has actually been injected with a virus of the pox family. The recipients are now know as vector’s. Through transfection the virus has been able to infect every single cell of the human body on such a nano scale that it’s undetectable until activated with a frequency, you guess what that may be but I think we all know what that will be. Studies regarding cDNA and transfecting vector’s from the mid 90’s. If you didn’t take the injection your clear. My guess? Start high infection with low mortality. Fowlpox or Monkeypox. Then change the frequency and high mortality with smallpox. These scumbags have made my life a living hell since I came across their documents and admissions. It’s only fair to warn the world. Wish you all the best of luck.